Identification of Bacteria from Diarrheic foals in Punjab, Pakistan

Ikramul Haq1, Aneela Zameer Durrani*1, Muhammad Sarwar Khan1, Muhammad Hassan Mushtaq2, Imtiaz Ahmad3, Amjad Khan2 and Mehboob Ali1

1Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan

2Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore, Pakistan

3Faculty of Veterinary and Animal Sciences, University of the Poonch, Rawalakot, (AJK), Pakistan

ABSTRACT

Foal diarrhea in working equines is reported as the most common cause of mortality. Lahore and Sahiwal districts of Punjab, Pakistan were selected during 2016 for the present study. A total of 616 foals were examined out of which 447 diarrheic foals were sampled for isolation of bacteria. During the study 294 faecal samples (horse n=217, mules n=23 and donkey n=54) were diagnosed positive for bacteria. The overall prevalence of diarrhea was 72.56% with bacteria in 65.77% of diarrheic cases. The prevalence of different bacterial species shows that E. coli was the most prevalent bacteria with 48.77% as compared to Clostridium perfringens (18.56%) and Salmonella (17.9%). It was concluded that bacteria were the major cause of foal diarrhea in Punjab, Pakistan.

Article Information

Received 12 July 2017

Revised 02 September 2017

Accepted 07 November 2017

Available online 03 January 2018

Authors’ Contributions

AZD, MSK, MHM and IH designed the study. IH and AZD executed the experiment and analysed the samples. IA, AK and MA helped in data analysis and article drafting.

Key words

Foal diarrhea, Salmonella, Clostridium perfringens, E. coli

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.1.sc5

* Corresponding author: [email protected]

0030-9923/2018/0001-0381 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Almost 80% of the foals, experience at least one episode of diarrhea in their life (Frederick et al., 2009). The causes of diarrhea may be infectious or non-infectious. The infectious causes of diarrhea include Salmonella Spp., Escherichia coli, Clostridium perfringens and Rotavirus (Lester and Magdigen, 2009). Among bacterial causes of foal diarrhea Salmonellosis is considered as an important and deadly disease caused by different serotypes of Salmonella enterica as compared to any other bacterial infection (Hassenin et al., 2010a). Salmonella is reported to be more prevalent in astrointestinal ailments in foals (Hartmann et al., 1996). Along with Salmonella, E. coli is most commonly isolated bacterium from foals (Hassenin et al., 2010b). The other species of bacteria involved in the disease are Clostridium (Netherwood et al., 1996). Salmonellosis occurs in different clinical forms in horses resulting in severe diarrhea and fatal septicemia in foals (Kohn, 1982). It causes higher mortality as compared to any other bacterial infection. Studies have shown 13% prevalence of Salmonellosis in foals with gastrointestinal disease. The recovered animals may shed the organism for variable period of time. The infection occurs through contaminated food and water (Hartmann et al., 1996) .In India Salmonella were isolated from 6.77% of faecal samples of equines (Singh et al., 2007). S. typhimurium was found as common bacteria in horses (Rajasekhar and Babu, 1992). Different strains of entero-toxigenic E. coli were isolated from foals (Holland et al., 1996). The bacterium is considered virulent due to its ability of affecting epithelium of intestinal tract (Mainil et al., 1993; Fisher et al., 1994). E. coli produce enterotoxins that cause epithelial cells to secrete electrolytes and water profusely and results in diarrhea (Ooms and Degryse, 1986). Sporadic cases of diarrhea in foals were reported caused by different type of Clostridium (Netherwood et al., 1996). Isolation and identification of bacteria causing foal diarrhea is important as some bacteria are zoonotic (Tillotson et al., 1997). Data on prevalence of foal diarrhea caused by different types of bacteria is not available in Pakistan. This is the first study of its kind in the country because an active surveillance is prerequisite to determine the extent of bacterial causes of foal mortality.

Material and methods

The study was carried out in rural and urban areas of Lahore and Sahiwal districts of Punjab, Pakistan to record the prevalence of bacterial diarrhea in foals during year 2016.

A total of 447 freshly passed faecal samples (foals up to 1 year of age) were collected from diarrheic foals of different species (horse n=318; donkey n=82 and mule n=47) in sterilized labeled container having phosphate buffered saline. The samples were cultured for isolation of Salmonella, C. perfringens and E. coli with subsequent PCR for confirmation of microbiota involved.

All 447 faecal samples were extended in 10 ml buffered peptone water and incubated at 37°C for 18 h. After pre-enrichment, faecal samples were selectively enriched in tetrathionate broth (TTB) at 42°C for 24 h for salmonella. For C. perfringens, samples were enriched in anaerobically sterilized Brucella broth and for E. coli were enriched in brain-heart infusion (BHI). After 24 h, a loopful was taken from the enriched samples and were streaked on brilliant green agar plates and the cultured plates were incubated at 37 °C for 24 h for salmonella and E. coli (Singh et al., 2007). Clostridium perfringens agar base medium supplemented with egg yolk emulsion @ 50 ml/ liter and D-cycloserine @ 2 vials/litter was used to culture C. perfringens (Despina et al., 2010). Suspected colonies of Salmonella (transparent colonies with reddish periphery), E. coli (colorless) and C. perfringens (black color) were re-cultured to get pure culture (Singh et al., 2007).

DNA was extracted using commercially available DNA extraction Kit (QIAGEN, Germany, Qiagen Street 1, 40724 Hilden, Germany) accordinag to company guidelines and procedure.

Data from questionnaires were entered into Microsoft Excel spread sheet. Data was validated through crosschecking the computerized recorded data with those of original complete data copy. Statistical analysis of data was conducted through SPSS Version 20.00. Prevalence data were analyzed by applying Chi-square test.

Results

Salmonella colonies appeared as transparent colonies with reddish periphery, on brilliant green agar. The suspected colonies were re-cultured for purification and expressed same colony characteristics. E. coli showed yellowish green colonies on brilliant green agar and arepeated same characteristics after re-culture. C. perfringens showed black colonies surrounded by transparent zones in cultures in C. perfringens agar base medium supplemented with egg yolk emulsion @ 50 ml/ liter and D-Cycloserine @ 2 vials/ litters.

Using the genus specific primer, a product of 496bp was generated for Salmonella species. The bacteria were found involved in the disease independently in 36 horse foals and in 10 donkey foals (Table I). The bacteria were not found in mule foals as the only cause of infection. However, the bacterium was found in mixed bacterial infection in all three species of equines (Table I). A product of 405bp was generated in case of C. perfringens. The bacteria were found in 11 horse and 4 donkey foals. In mules, the bacterium was involved in mixed infections. The E. coli infection was conformed in 112 horses, 9 mules and 32 donkey foals as a sole cause of bacterial diarrhea (Table I). A 200bp product was generated in PCR.

An overall prevalence of foal diarrhea in two geographical locations of Punjab was 72.56%. The overall bacterial cause of diarrhea was found 65.77% among diarrheal foals. The prevalence of diarrhea in horse, mule and donkey foals was 72.93, 66.19 and 75.22% respectively (Table II). The bacterial diarrhea in three species revealed 68.55, 48.93 and 65.85% disease, respectively. Data on gender (in case of mule the appearance of the foal was considered) revealed 68.91% in male and 75.93% in female foals. The bacterial infection was found 58.82% in male 72.01% in female among diarrheal animals. Area wise prevalence was 72.58 and 72.54% in Lahore and Sahiwal districts. The bacteria were isolated from 45.48 and 50% of the diarrheal animals. Chi-square analysis showed significant difference (p<0.05) between male and female animals in overall disease in foals. The data on bacterial foal diarrhea revealed significant difference among species, gender and area (Table II). The bacteria were found in 294 animals as mixed and single infection. The prevalence of different bacterial species show that E. coli was the most prevalent bacteria with 48.77% as compared to C. perfringens (18.56%) and Salmonella (17.9%) (Table II). The bacteria were isolated as infection with single specie or mixed infection with other bacteria. The data showed that 80 foals were infected with 2 or more than 2 bacteria. Chi-square analysis showed significant difference (p<0.05) except area wise prevalence of single or mixed bacterial infection (Table I).

Discussion

Foal diarrhea was the major reported cause of mortality in early age (Baverud et al., 2003). The diagnosis was made through different techniques. The culture of bacteria and subsequent confirmation through PCR was performed by many researchers (Singh et al., 2007; Walker et al., 1995) amplification of DNA (PCR) was reported as efficient method of confirmation (Amavisit et al., 2001; Ward et al., 2005). The prevalence rate of 80% diarrhea up to six months (age) was reported by (Frederick et al., 2009; Dunkel and Wilkins, 2004). The findings of the present study were in line with these reports. The findings of the present study were in agreement with previous Reported studies that showed that the major bacterial species involved in foal diarrhea were Salmonella species (Fredrick et al., 2009), C. perfringens (Netherwood et al., 1996; Weese et al., 2001; Tillotson et al., 2002; Fredrick et al., 2009) and E. coli (Hamzah et al., 2013; Quinn et al., 2002). These three species were found in foals of horse, mule and donkey in both areas of Punjab. In the present study, E. coli was found the most prevalent specie of bacteria. The findings are in agreement with earlier study conducted by Hamzah et al. (2013) who reported E. coli 48.75% and 44.00%, respectively. The higher prevalence of the E. coli may due to the presence of the bacteria in equine gastrointestinal tract as commensal organism Quinn et al. (2002). The prevalence

Table I.- Species of bacteria isolated from Diarrheic foals in different district of Punjab, Pakistan.

Table II.- Prevalence of bacteria in Diarrheic foals in Lahore and Sahiwal districts of Punjab, Pakistan.

of E. coli in male foals was 47.05 % while in female foal it was isolated from 50.02% of foals. Same trend was shown by the study conducted by Harris et al. (2012). In our study the prevalence of E. coli was more in horses (49.37%) as compared to mules (48.9%) and donkey foals (46.34%). Quinn et al. (2002), Hamzah et al. (2013) reported similar findings. In the present study, Salmonella was isolated from 17.9% of the samples. Our findings are in line with the earlier studies in which salmonella was documented as important cause of equine diarrhea, Singh et al. (2007) reported 13% and Fredrick et al. (2009) 12% Salmonella in foals. The study conducted in India having similar geographic conditions showed 6% prevalence of salmonella in equines using culture technique and 12.5% with PCR (Singh et al., 2007). The study also agrees with earlier report that showed 1% to 17% prevalence of salmonella from faecal sample or rectal swabs (Traub-Dargatz et al., 2000; Babu, 2003). The results showed that the occurrences of salmonella were significantly associated with the gender of the foals. The prevalence was high in females (20.1%) as compared to males (14.21%). Our results were not with in agreement with pervious study conducted by Harris et al. (2012). Species wise prevalence of salmonella in our study showed higher percentage in horses than donkeys. The results are concurrence with earlier study conducted by (Singh et al., 2007). In the present study, C. Perfringens was isolated and confirmed from 18.56% of the diarrheic foals. The results of the study were in agreement with the pervious study by Fredrick et al. (2009). The bacteria were found significantly involved in foal diarrhea (Netherwood et al. 1996). Tillotson et al. (2002) reported that 85% of isolates were type A and were found positive for entero toximic genes. The study is not in agreement with previous study in which C. perfringens was isolated from 90% of foals from 6 farms and reported non-significant association between the isolation of bacteria and the occurrences of diarrhea (Tillotson et al., 2002). The result showed that prevalence of Clostridium Perfirengenes was 18.8% in females and was 16.86% in male foals. The results of our study showed same trend as reported by earlier study conducted by Harris et al. (2012). The prevalence of C. perfringens in different species of equines showed that the prevalence was high in horses (18.86%) as compared to donkeys (14.74%). Earlier reports showed 18% prevalence of C. perfringens in horses (Fredrick et al., 2009) and 90% in diarrheic horse foals (Tillotson et al., 2002). The bacterial diarrhea in female foals was significantly higher than males. Contrarily Singh et al. (2007) reported more bacterial infection in male foals. This difference in findings might be due to the difference in management or sample size of the two studies. The difference between male and female foals may be attributed to the physiology and stress difference of gender (Carter et al., 1986). The disease was reported significantly high in mules followed by horses and donkeys (Singh et al., 2007; Singh et al., 2005) due to higher susceptibility of intestine to colonization of bacteria due to exposure to unhygienic conditions in mules. Losinger et al. (2002) also reported more prevalence in mule with the view that mules are grouped for draught purpose. These studies were reported in adult animals. The data of the present study revealed higher prevalence in horse foals (68.23%) followed by donkeys (65.85%) and mules (48.93%). The difference may attribute to the age and number of samples. During the present study, higher prevalence of bacterial diarrhea was found in Sahiwal district (50%). The trend of the disease was found almost similar between two regions.

It can be concluded that isolation of E. coli, Salmonella and C. perfringens from foals of Lahore and Sahiwal districts of Punjab, Pakistan indicated that the bacteria are prevalent in these districts as intestinal microbiota since early ages. Further investigation from different geographical region of country are pre-requisite to equines foals.

Acknowledgement

The authors are highly obliged to Higher Education Commission (HEC) Pakistan for funding the research under Project No. 4263; Titled: “Epidemiology of Major Viral, Bacterial and Parasitic Causes of Foal Diarrhea” The authors also appreciate the cooperation of RVFC in field work of the project.

Statement of conflict of interest

Authors have declared no conflict of interest.

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